The present invention relates generally to a novel technology for conjugating amphiphilic oligomers and polymers to peptides in order to modulate their pharmacokinetic profile and thereby improve their clinical utility. The conjugates of the invention have the ability to stabilize and deliver luminal cholecystokinin releasing factor (LCRF) to receptors in the gut, without absorption in the bloodstream.
Endogenous LCRF acts at receptors on the luminal surface of gut epithelial cells, causing them to release cholecystokinin (CCK), a polypeptide hormone that induces satiety and reduces food intake, into the bloodstream. Exogenous LCRF, stabilized and delivered to the gut, will mimic normal physiological activity. The chemical modification of peptides regulating feeding behavior has the potential to treat obesity, a serious and growing public health problem in all industrialized nations, especially the United States.
Obesity is near epidemic proportions in industralized countries, and its prevalence is increasing. The pathogenesis of obesity is complex, involving the interaction of lifestyle, dietary, behavorial and genetic factors. It is an object of the present invention to provide a drug that will induce satiety and will thereby produce weight loss.
The role of neuro- and gastric-peptides in regulation of feeding has been a major focus of obesity research. Convincing evidence exists that CCK inhibits feeding. (Schwartz, G. J., Berkow, G., McHugh, P. R., Moran, T. H., xe2x80x9cGastric Branch Vagotomy Blocks Nutrient and Cholecystokinin-induced Suppression of Gastric Emptying,xe2x80x9d Am. J. Physiol., 264:R630-7 (1993) (the disclosure of which is incorporated herein in its entirety)). Regulation of CCK expression is achieved in large part by regulation of LCRF, which is constitutively expressed in the duodenum in rats. In humans, it is postulated that stimulation by nutrients is required to stimulate secretion of the putative CCK-releasing factor. (Liddle, R. A. xe2x80x9cCholecystokinin Cells,xe2x80x9d Ann. Rev. Physiol., 59:221-42 (1997) (the disclosure of which is incorporated herein in its entirety)). After a protein meal, proteins in the food saturate the available trypsin, leaving a greater amount of LCRF unhydrolyzed, which then binds to the CCK cells. This causes them to release CCK, leading to satiety.
In one aspect, the conjugates of the present invention can be used to target the LCRF receptor with LCRF, its natural agonist, in order to initiate CCK release.
It is an object of the invention to provide a means for delivering LCRF to its target receptor. This object is achieved by conjugation of PEG oligomers or polymers modified with alkyl groups to proteins and peptides, to improve their pharmacologic properties. The present invention uses amphiphilic oligomer and polymer conjugation to vary the hydrophobicity and hydrophilicity of drug molecules. This reduces immunogenicity, prevents enzymatic degradation and facilitates oral delivery and partitioning to various tissues.
It is another object of the invention to provide conjugated LCRF which will be able to induce CCK release from CCK-releasing cells, leading to satiety and a reduction in food intake. We propose to deliver LCRF using our proprietary amphiphilic polymer conjugation technology, which will protect LCRF from proteolysis and confine it to the lumen of the gut for an extended period of time, producing satiety. We propose that such a conjugated LCRF would be an effective and safe therapeutic for chronic treatment of obesity.
The invention also provides synthetic methods for attaching an amphiphilic polymer to the N-terminal residues of LCRF, a 35 amino acid fragment that exhibits all the biological activity of full-length LCRF.
The amphiphilic conjugate comprises a polyethylene glycol (PEG) moiety and an alkyl chain. The alkyl chain can integrate into membranes on the epithelium of the intestine, bringing the conjugate in close proximity to LCRF receptors, which are located on the CCK-releasing cell surface. Stability of the peptide will be prolonged to maintain bioactivity. Moreover, where greater stability and a reduced tendency to penetrate the intestinal epithelium is required, peptides can be provided with conjugates at the N-terminus, at K19 and the C-terminus.
The operability of the LCRF conjugate molecules can be validated in a cell-based assay, using freshly prepared CCK-releasing cells obtained from rat intestine. A radioimmunoassay (RIA) can be used to detect CCK release.
LCRF is secreted in the duodenum and is physiologically regulated by proteolysis, particularly by trypsin. By protecting LCRF from proteolytic digestion, it will retain activity, bind to, and activate, LCRF receptors on CCK cells.
An amphiphilic polymer can be covalently attached to the xcex5 amino group of K191, adjacent to the only trypsin cleavage site in LCRF (1-41), thus protecting LCRF from trypsin proteolysis by steric hindrance. It is preferable that modifications made to LCRF must not obstruct key residues involved in receptor binding. Since it is known that K19 is within the region crucial for receptor binding, it is preferable to attach the K19 conjugate with a linker that is slowly hydrolyzed under conditions found in the duodenum. As the K19 conjugate is released, the peptide then regains full activity. Slow hydrolysis may also extend action of the delivered LCRF, to minimize dosing.
About 55% of the US population is overweight or obese, with serious public health consequences. (The National Heart Lung and Blood Institute in cooperation with The National Institutes of Diabetes and Digestive and Kidney Diseases, xe2x80x9cClinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults,xe2x80x9d NIH Publication no. 998-4083 (1998) (the disclosure of which is incorporated herein in its entirety)). Three of the most serious sequelae of obesity are heart disease, hypertension, and diabetes. Dieting and exercise have been largely unsuccessful long-term strategies for most overweight pepole (Wadden, T. A. xe2x80x9cTreatment of Obesity by Moderate and Severe Caloric Restriction, Results of Clinical Research Trials,xe2x80x9d Ann. Intern. Med., 119:688-693 (1993) (the disclosure of which is incorporated herein in its entirety)); thus there is a need for pharmaceuticals that suppress appetite. One approach to appetite suppression is to induce release of CCK, a peptide hormone that produces satiety and reduces feeding.
CCK is one component of the hormonal system that tightly regulates hunger and satiety, digestion, and disposal of nutrients. CCK has several activities: it induces satiety and reduces food intake; stimulates gallbladder contraction; increases pancreatic enzyme secretion; and delays gastric emptying. When food, especially fat and protein, enters the small intestine, CCK is released into the blood where it binds to receptors in the peripheral nerves, pancreas, gall bladder, and stomach. (Mutt, V. xe2x80x9cSecretin and Cholecystokinin,xe2x80x9d in xe2x80x9cGastrointestinal Hormones,xe2x80x9d 251-320 (ed. V. Mutt, San Diego, Academic Press, 1998) (the disclosure of which is incorporated herein in its entirety)).
CCK release in the intestine is under negative feedback regulation. (Green, G. M. and Lyman, R. L. xe2x80x9cFeedback Regulation of Pancreatic Enzyme Secretion as a Mechanism for Trypsin Inhibitor-induced Hypersecretion in Rats,xe2x80x9d Proc. Soc. Exp. Biol. Med., 140:6-12 (1972) (the disclosure of which is incorporated herein in its entirety)). LCRF is a constitutively produced, trypsin-sensitive intestinal CCK-releasing peptide responsible, in part, for this negative feedback phenomenon. (Lu, L., Louje, D., and Owyang, C. xe2x80x9cA Cholecystokinin Releasing Peptide Mediates Feedback Regulation of Pancreatic Secretion,xe2x80x9d Am. J. Physiol., 256:G430-G435 (1989); Miyasaka, K., Guan, D., Liddle, R. A. and Green, G. M. xe2x80x9cFeedback Regulation by Trypsin: Evidence for Intraluminal CCK-releasing Peptide, Am. J. Physiol., 257:G175-G181 (1989) (the disclosure of which is incorporated herein in its entirety)). FIG. 1 (adapted from Miyasaka K and Funakoshi A (1998) Pancreas 16:279) summarizes the regulation of CCK release by LCRF. In the basal or fasting state, all LCRF is rapdily cleaved by trypsin. Upon ingestion of protein or trpysin inhibitors, some LCRF remains intact and binds to the CCK-releasing cell, causing CCK release and satiety. When LCDF is infused into the duodenum of rats with trypsin inhibitors, it causes a reduction of food intake. (Green, G. M., Taguchi, S., Friestman, J., Chey, W. Y., Liddle, R. A. xe2x80x9cPlasma Secretin, CCK, and Pancreatic Secretion in Response to Dietary Fat in the Rat,xe2x80x9d Am. J. Physiol., 256:G1016-1021 (1989) (the disclosure of which is incorporated herein in its entirety)).
LCRF is a Potent CCK Releasing Factor. LCRF is one of at least three peptides responsible for causing CCK cells to release CCK into the bloodstream. LCRF is the most potent of these peptides. Two other peptides secreted into the intestines are also found to stimulate CCK release: diazepan binding inhibitor and monitor peptide. The latter controls CCK release by positive feedback regulation. However, the potency of these two peptides for stimulating CCK release is less than LCRF (down 100-1,000 fold and 6 fold respectively). (Liddle, R. A., unpublished results; H. Spannagel A. W., Reeve, J. R., Liddle, R. A., Guan, D., Green, G. M. xe2x80x9cAn Amino-terminal Fragment of LCRF, LCRF (1-35), Has the Same Activity as the Natural Peptide,xe2x80x9d Am. J. Physiol., 273:G754-758 (1997) (the disclosures of which are incorporated herein in their entireties)).
The nature of the receptor-LCRF interaction has not been established, as the receptor has not been identified. However, there is strong evidence that the receptor resides on the cell surface. Dr. Rodger Liddle of Duke University, our collaborator, has demonstrated that LCRF stimulates CCK release from intestinal endocrine cells through a calcium influx pathway. (Liddle, R. A., Prpic, V., Wang, Y., Romac, J., Green, G. M., Reeve, J. R. xe2x80x9cLuminal Cholecystokinin Releasing Factor (LCRF) Stimulated CCK Release from Intestinal Endocrine Cells through a Calcium Influx Pathway,xe2x80x9d Gastroenterology, 116:A622 (1999) (the disclosure of which is incorporated herein in its entirety)). Increase in intracellular calcium are a typical signaling mechanism for receptor signaling and strongly suggest that the receptor resides on the cell surface.
Feeding of trypsin inhibitors or diversion of bile-pancreatic juice stimulates pancreatic exocrine secretion and CCK release in rats and humans. (Slaff, J. D., Jacobson, C. R., Tulman, C., et al., xe2x80x9cProtease-specific Suppression of Pancreatic Exocrine Secretion,xe2x80x9d Gastroenterology, 87:44-52 (1984) (the disclosure of which is incorporated herein in its entirety)). A CCK-stimulating peptide that is sensitive to tryptic digestion was first purified from rat intestinal washings. (Spannagel, A. W., Green, G. M., Guan, D., Liddle, R. A., Faull, K. and Reeve, J. R. xe2x80x9cPurification and Characterization of a Luminal Cholecystokinin-releasing Factor from Rat Intestional Secretion, Proc. Natl. Acad. Sci. USA, 93:4415-4420 (1996) (the disclosure of which is incorporated herein in its entirety)). This peptide, named luminal cholecystokinin releasing factor (LCRF), stimulates CCK release from isolated rat, mouse, and human intestinal cells and a cell line derived from an intestinal endocrine tumor (STC-1 cells). (Rindi, G. S., Grant, S. G. N., Yiangou, Y., et al. xe2x80x9cDevelopment of Neuroendocrine Tumors in the Gastrointestinal Tract of Transgenic Mice. Heterogeneity of Hormone Expression,xe2x80x9d Am. J. Pathol., 136:1349-1363 (1990) (the disclosure of which is incorporated herein in its entirety)). The 35 residue N-terminal fragment of LCRF has all of the biological activity of the native peptide, which is between 60-65 residues long (8136 Da). Using synthetic peptides, it has been found that residues 11-25 are responsible for the CCK releasing properties of LCRF, producing about 60% of the activity of the 1-41 fragment. (Spannagel, A. W., Reeve, J. R., Jr, Greeley, G. H., Jr, Yanaihara, N., Liddle, R. A., Green, G. M. xe2x80x9cBioactivity of Intraduodenally and Intravenously Infused Fragments of Luminal Cholecystokinin Releasing Factor (LCRF),xe2x80x9d Regulatory Peptides, 73:161-4 (1998) (the disclosure of which is incorporated herein in its entirety)). However, the gene for LCRF has not been identified, and as of December 1999, no homologues exist in publicly available protein or DNA sequence databases.
The present invention provides an LCRF conjugate that retains the capacity to bind to the LCRF receptor on CCK cells; is protected from proteolytic cleavage; and is confined to the lumen of the gut by virtue of its molecular weight. LCRF receptor binding will cause the CCK cells to release CCK into the blood stream, leading to satiety and a reduction in food intake. Accordingly, the present invention provides LCRF conjugates that provide an effective orally delivered treatment of obesity.
Practical limitations exist to the use of peptides as drugs. Proteolysis, both in the gut and in the bloodstream, is a major barrier to using peptides as therapeutics. Indeed, the transient nature of many endogenous peptides, including LCRF, is a feature of their regulatory function. Another difficulty encountered with non-endogenous peptides is immunogenicity. As a result of these problems, the approach of the pharmaceutical industry has been to create small, non-peptide molecules using medicinal chemistry. And while medicinal chemistry has been successful, it is very time consuming. Furthermore, chemical drugs may have unexpected toxicity or teratogenicity.
While the use of xe2x80x9cPEGylatedxe2x80x9d proteins is well established, to date they have been confined to injectable use. The present invention provides orally available conjugates of polypeptides, such as insulin, leu-enkephalin, and calcitonin. (Ekwuribe, N., Ramaswamy, M., Allaudeen, H. S., Rajagopalan, J. S., et al., xe2x80x9cOral Insulin Delivery: Hydrolyzable Amphiphilic Oligomer Conjugates Prolong Glucose Reduction,xe2x80x9d Proceed Intl. Symp. Control Release Bioactiv. Materials,xe2x80x9d Abstract 242 (1999); Anderson, W. R., Ekwuribe, N., Ansari, A., Harris, T. M., Surguladze, D. xe2x80x9cStructure Activity Relationship Assessment of Conjugated Enkephalins in Centrally Mediated Analgesia,xe2x80x9d Soc. for Neuroscience, Abstracts 25(1):180 (1999); Krishnan, D. R., Ramaswamy, M., Rajagopalan, J. S., Anderson, W. R., Allaudeen, H. S., Myung, S., Ekwuribe, N. xe2x80x9cOral Delivery of Calcitonin by Conjugation with Amphiphilic Oligomers,xe2x80x9d Proceed. Intl. Symp. Control Release Bioactiv. Materials, Abstract 243(1999) (the disclosures of each of which are incorporated herein in their entireties)). Specifically, present invention provides conjugates comprising PEG units linked to alkyl chains. (U.S. Pat. No. 5,681,811: Conjugation-stabilized Therapeutic Agent Compositions, Delivery and Diagnostic Formulations Comprising Same, and Method of Making and Using the Same (the disclosure of which is incorporated herein in its entirety)). By adjusting the number of PEG monomers, the type and length of the alkyl chain and the exact nature of the PEG-peptide linkage, desired properties of lipophilicity can be tailored to a specific use. In addition, the exact nature of the PEG-peptide linkage can be varied such that it is stable or sensitive to hydrolysis at physiological pH. The hydrolyzable linker allows the intact, unconjugated peptide to be released over time, to act as a prodrug.
Cell-based assays can be used to validate to capacity of amphiphilic oligomer-LCRF conjugates for oral delivery in animals. Animal studies can then be used to validate the appetite-suppressing ability of the conjugates. For example, rats can be subjected to behavioral studies to assess the appetite suppression profile of the LCRF-conjugate. If the conjugate shows desired ability to suppress appetite in rats, the compound will be subjected to detailed pharmacokinetic, and pharmacodynamic studies in rats, as well as toxicity testing.
An enormous effort is being made to develop pharmaceuticals for the treatment of obesity. A recently approved drug for obesity is the lipase inhibitor orlistat, which prevents fat uptake, but does not affect appetite. Orlistat (Xenical) may lead to diminished fat-soluble vitamin intake and can have very unpleasant side effects. Marketed appetite suppressants include sibutramine (Meridia), a mixed neurotransmitter reuptake inhibitor, and sympathomimetic agents. However, use of these drugs is limited by CNS effects. In 1997, the appetite suppressant drug combination fenfluramine and dexfenfluramine (Fen-Phen) was found to cause cardiac valvulopathy and was withdrawn from the market. (U.S. Department of Health and Human Services, xe2x80x9cCardiac Vavulopathy Associated with Exposure to Fenfluramine and Dexfenfluramine: U.S. Department of Health and Human Services Interim Public Health Recommendations,xe2x80x9d Morb. Mortal. Wkly. Rep., 46:1061-1066 (1997) (the disclosure of which is incorporated herein in its entirety)). Neuropeptide Y antagonists, kappa opioid receptor antagonists, melanocortin-4 agonists and beta-3 adrenergic receptor antagonists are all in preclinical development for appetite suppression. Leptin and neuropeptide Y, two endogenous peptides, are involved in controlling the long-term regulation of food consumption and energy expensiture. (Friedman, J. M. Halaas, J. L. xe2x80x9cLeptin and the Regulation of Body Weight in Animals,xe2x80x9d Nature, 395:763-770 (1998) (the disclosure of which is incorporated herein in its entirety)). While these two peptides are being studied intensely, neither has proven successful in clinical studies.
The CCK pathway is involved in the modulation of feeding patterns, rather than long-term weight maintenance. (Spiegelman, B. M., Flier, J. S. xe2x80x9cAdipogenesis and Obesity: Rounding Out the Big Picture,xe2x80x9d Cell, 87:377-389 (1996) (the disclosure of which is incorporated herein in its entirety)). Three of the approaches to discovery and development of drugs for obesity directly target the CCK pathway, including CCK-A agaonists, inhibitors of CCK proteolysis, and agonists to the LCRF receptor.
Several benzodiazepine analogs and peptidomimetics are in preclinical development as CCK-A agonists. These compounds have been shown to decrease food intake in rats and are in preclinical development for the treatment of obesity by Abbott Laboratories and Glaxo Wellcome. Further validation of this approach is the use of CCK-A antagonists as a treatment for anorexia nervosa.
Butabindide is a cholecystokinin-inactivating peptidase inhibitor that is undergoing preclinical investigation by INSERM, France, for the treatment of obesity. However, the duration of action of butabindide is too brief for clinical utility, and investigation of other analogs is underway.
Synthetic small molecules that are LCRF receptor against could be used to stimulate CCK release. However, structure-based drug design is not a currently viable strategy because of lack of structural data on either LCRF or its receptor.
By 2005, the number of people dieting to lose weight for health reasons is expected to be 5.8 million. The total market for weight-loss products and services, including diet foods, in the United States for health or cosmetic reasons is about $30 billion. Despite the paucity of safe and effective products, Americans spent $243 million on obesity drugs in 1998. Clearly, the market for a safe, effective and chronic treatment for obesity is tremendous.
The present invention provides a composition comprising a protein or peptide, such as LCRF, coupled with one or more molecules of a non-naturally-occurring polymer, said polymer comprising a lipophilic moiety and a hydrophilic polymer moiety, thereby imparting balanced lipolhilic and hydrophilic characteristics to the composition such that the composition is soluble in pharmaceutically acceptable solvents and able to interact with biological membranes.
The present invention provides a composition comprising a protein or peptide, such as LCRF, coupled with one or more molecules of a non-naturally-occurring polymer, said polymer comprising: (i) a lipophilic moiety; and (ii) a hydrophilic polymer moiety, thereby imparting balanced lipolhilic and hydrophilic characteristics to the conjugate such that the conjugate is soluble in pharmaceutically acceptable solvents and able to interact with biological membranes.
The present invention provides a composition comprising a protein or peptide, such as LCRF, coupled with one or more molecules of a non-naturally occurring polymer comprising a lipophilic moiety and a hydrophilic moiety wherein the composition is soluble in aqueous solvents and the LCRF is active in prophylaxis or treatment of obesity.
The present invention provides a composition comprising a protein or peptide, such as LCRF, covalently coupled with one or more molecules of a polymer comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the physiologically active peptide, the linear polyalkylene glycol moiety and the lipophilic moiety are conformationally arranged in relation to one another such that the LCRF in the LCRFcomposition has an enhance in vivo resistance to enzymatic degradation, relative to the LCRFalone.
The present invention provides a composition comprising a triglyceride backbone moiety, having: LCRF covalently coupled with the triglyceride backbone moiety through a polyalkylene glycol spacer group bonded at a carbon atom of the triglyceride backbone moiety; and at least one fatty acid moiety covalently attached either directly to a carbon atom of the triglyceride backbone moiety or covalently joined through a polyalkylene glycol spacer moiety.
The present invention provides polysorbate complex comprising a polysorbate moiety including a triglyceride backbone having a fatty acid group covalently coupled to one of the xcex1,xcex1xe2x80x2 and xcex2 carbon atoms thereof, and having a polyethylene glycol group covalently coupled to one of the xcex1,xcex1xe2x80x2 and xcex2 carbon atoms thereof.
The present invention provides a stable, aqueously soluble, conjugated LCRF complex comprising a LCRF stabilizingly and conjugatively coupled to a polyethylene glycol modified glycolipid moiety.
The present invention provides a polysorbate complex comprising a polysorbate moiety including a triglyceride backbone having covalently coupled to carbon atoms independently selected from xcex1,xcex1xe2x80x2 and xcex2 carbon atoms thereof, functionalizing groups including a fatty acid group; and a polyethylene glycol group having a physiologically active moiety covalently bonded thereto.
The present invention also provides an oral administration dosage form for the treatment of obesity, comprising a pharmaceutically acceptable carrier and a stable, aqueously soluble, conjugated LCRF complex comprising LCRF coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
The present invention also provides a method of treating obesity in a human or non-human mammalian subject exhibiting such deficiency, comprising orally administering to the subject an effective amount of a conjugated LCRF composition comprising a stable, aqueously soluble, conjugated LCRF complex comprising LCRF covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
The present invention provides a method of prophylactically or interventionally treating potential or developed obesity in a human or non-human mammalian subject LCRF, comprising administering to the subject an effective amount of a conjugated LCRF composition comprising a stable, aqueously soluble, conjugated LCRF complex comprising LCRF coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
The present invention provides a method of prolonging the activity of LCRF in an in vivo or in vitro system, comprising conjugatively coupling LCRF with one or more molecules of a non-naturally-occurring polymer comprising a lipophilic moiety and a hydrophilic polymer moiety to yield a conjugatively coupled polymer-LCRF composition, and introducing the conjugatively coupled polymer-LCRF composition to thein vivo or in vitro system.
The present invention also provides, in a preferred embodiment, a compound of the formula: 
where n is from 3 to 230 and m is from 0 to 20.
The present invention also provides, in a preferred embodiment, a compound of the formula: 
where n is from 3 to 230 and m is from 0 to 20 and X is selected from the group consisting of N, O or S.
The present invention also provides, in a preferred embodiment, a compound of the formula: 
where n is from 3 to 230 and m is from 0 to 20, where the protein is preferably a therapeutic protein.
The present invention also provides, in a preferred embodiment, a compound of the formula: 
where n is from 3 to 230 and m is from 0 to 20 and X is selected from the group consisting of N, O or S, where the protein is preferably a therapeutic protein.